Human mesenchymal stem cells (hMSCs) havebeen demonstrated to exhibit characteristic cytosolic Ca2+ ([Ca2+]i) oscillations in response to physicochemical factors,including soluble factors, matrix proteins and microenvironment.It has been postulated that the calciumoscillation at the early stages of hMSC differentiation mayserve as a biophysical predictor. The goal of the presentstudy is therefore aimed at elucidating the matrix proteinmediated[Ca2+]i oscillation that occurs in the early stagesof hMSC osteogenic differentiation. On the collagen substratesof different stiffness and concentrations, intracellular[Ca2+]i oscillations of hMSCs exhibited rapid spikeson the hard substrates and the opposite on the soft substrate.Cells seeded on a hard substrate also expressed significantly high levels of osteogenic gene markers, suggestinga key role of intracellular calcium in the transductionof ECM signaling to the hMSC differentiation. Our findingsrevealed that the characteristics of [Ca2+]i oscillationare regulated by the biochemical and mechanical propertiesof the collagen substrate. Furthermore, β1-integrinand associated signaling proteins found in focal adhesions(FAs) were involved in the signaling pathways. Becausethe [Ca2+]i signaling and stemcell differentiation appearclosely correlated, the regulation of [Ca2+]i signalingthrough the modification of engineeredECMmay provide acontrollable exogenous technique to direct the fate of hMSCs.